Visualizing Motor Neuron Projections and Axon Branching Using Light Sheet Fluorescence Microscopy

Take a chemically fixed and cleared tissue segment from a transgenic mouse embryo expressing green fluorescent protein (GFP) in spinal motor neurons.

Mount the tissue vertically onto a modified pipette tip.

Attach the tip to a capillary secured within the light sheet fluorescence microscope.

Place a buffer-filled sample chamber beneath the tissue to immerse it and clear debris.

Using imaging software, align the tissue in the light path.

Begin image acquisition. 

The light source emits a thin sheet of laser light, exciting GFPs in a single plane of the tissue to emit fluorescence.

A detector positioned perpendicular to the light path captures the emitted fluorescence.

Acquire images of the spinal motor neurons to generate a 3D reconstruction of the axonal branching morphology.

Use software to map axons, identifying branching patterns and endpoints to assess motor neuron growth and connectivity.